Enteropathogenic Escherichia coli (EPEC) are an important cause of diarrhea in infants. The long-term objectives of this project are to understand the pathogenesis of disease due to this organism. Previous work on this project has resulted in the discovery of a 35 kb pathogenicity island called the Locus of Enterocyte Effacement (LEE) that is responsible for the attaching and effacing (AE) histopathology on intestinal epithelial cells that is the hallmark of EPEC infections. The LEE island encodes the epithelial cell adhesin named intimin, a type III protein secretion system, and several effector proteins that are translocated into epithelial cells to mediate the AE lesion. Typical EPEC strains possess the LEE plus the EAF plasmid that encodes a type IV pilus (BFP) that contributes to epithelial cell adherence and a positive regulator of EPEC virulence factors called Per. Typical EPEC strains are an important cause of infant diarrhea in developing countries but are infrequent causes of disease in the U.S. Atypical EPEC strains contain the LEE but lack the EAF plasmid. In contrast to typical EPEC strains, atypical EPEC appear to cause outbreaks and sporadic disease in adults in industrialized countries such as the U.S., Finland, and Japan. Recent studies of atypical EPEC in children with diarrhea in the U.S. suggest that the incidence of infections with this pathogen may equal or exceed that of Salmonella, Shigella, Campylobacter, or E. coli O157:H7. The proposed research goals for the next period of support include continued characterization of pathogenic mechanisms of typical EPEC strains, particularly genes encoded outside the LEE, and a new focus on potential pathogenic mechanisms of atypical EPEC. The specific aims are 1) Characterize the Long Polar Fimbriae (LPF) of EPEC and assess in vivo expression of this and other potential adhesins; 2) Identify and characterize genes regulated by the Ler and Per regulators of EPEC virulence factors using genomic arrays; 3) Characterize human host epithelial cell transcriptional responses to EPEC infection using in vitro organ culture (IVOC); and 4) Characterize potential virulence factors in atypical EPEC strains.